Project Summary Cardiovascular disease (CVD) is the major cause of death in the US, which has resulted in an intense search for CVD risk factors. Recent efforts have focused on novel CVD risk factors, as conventional risk factors may explain <50% of CVD. Endothelial progenitor cells (EPCs) are one such novel CVD risk factor. Endurance exercise training increases EPC number; however, most previous studies assessed only EPC numbers and colony forming units, which represent a minimal characterization of EPC function. This proposal expands on our previous work to study the cellular/molecular mechanisms underlying training-related changes in ex vivo EPC function, using a cross-sectional comparison combined with exercise training in sedentary older adults and training cessation in older endurance-trained athletes. We also will determine whether these training- induced changes translate to improved in vivo endothelial function. To better characterize ex vivo EPC function we will measure NADPH oxidase activity and expression, nitric oxide (NO) and reactive oxygen species (ROS) levels, and tube formation in CD34+-derived putative EPCs, under both standard and atherogenic culture environments. Our Hypothesis #1 is, that on a cross-sectional basis, older athletes will have better ex vivo EPC function under standard culture conditions and in response to an atherogenic culture environment than matched older sedentary individuals. Our definition of better EPC function is lower ex vivo EPC NADPH oxidase expression/activity and ROS levels and higher NO levels and tube formation. We will also assess in vivo endothelial function to determine if it can be used as a clinical index of ex vivo EPC function. Our Hypothesis #2 is that exercise training in sedentary individuals will improve, and training cessation in older athletes will worsen, ex vivo EPC function under standard culture conditions and in response to an atherogenic environment and in vivo endothelial function. These results will allow us to validly conclude that exercise training improves ex vivo EPC and in vivo endothelial function. We will then perform mechanistic studies by manipulating NADPH oxidase activity and NO levels in cultures to assess the degree to which EPC function in sedentary subjects can be improved by mimicking the intracellular EPC milieu evident in athletes. Our Hypothesis #3 is that both inhibiting NADPH oxidase and increasing intracellular NO in putative EPCs from sedentary individuals and from athletes after stopping training will improve ex vivo EPC function to levels similar to those of trained athletes. This mechanistic approach will allow us to quantify the extent to which exercise training effects on EPC function are a function of changes in EPC NADPH oxidase activity. Very few studies have addressed the interaction between EPCs, as a novel CVD risk factor, and exercise training, and virtually no studies have utilized human subjects and extended the studies to the cellular, metabolic, and mechanistic levels. Thus, we are in a unique position to expand dramatically our understanding of the mechanisms that underlie the benefits of exercise training on this novel CVD risk factor.